A major goal of this proposal is to develop a novel experimental paradigm to engineer preventative HIV-1 vaccines. The V1/V2 domain of HIV-1 envelope is an important target for vaccine development for several reasons. V2 interaction with the ?4?7 integrin of mucosal T cells may play a key role in the efficient capture of HIV-1 virus at the site of initial exposure during sexual transmission. The V1/V2 domain is also a key player in regulating neutralization sensitivity of HIV-1, and consists of epitopes recognized by some of the most potent broadly neutralizing antibodies reported to date. Here, we hypothesize that induction of antibodies by vaccination with V1/V2 immunogens that interferes, specifically with the V2 ?4?7 interactions, confers protection against HIV-1 acquisition. Since the conformation of V2 is highly variable, the V2 domain will be constrained by transplanting it into the compactly folded 9 kDa small outer capsid protein (Soc) from bacteriophage T4. To select for the functionally important V2 conformations, libraries of V2 domain variants consisting of founder/acute virus sequences, different glycosylation patterns, and random mutations will be constructed and expressed in CHO cells as secretory proteins. High throughput assays will be developed, one to select ?4?7 binding conformations and another to select V2 conformational antibody binding variants. The biologically active Soc-V2 conformational variants will be arrayed on phage T4 nanoparticles by incubating the purified Soc-V2 proteins with Soc-minus Hoc-minus phage, up to 870 copies per particle. A Hoc- fused targeting ligand such as Dec205 mAb will also be assembled on the same capsid, up to 155 copies per capsid, to target the T4-V2 nanoparticles to the antigen-presenting dendritic cells. The immunogenicity of T4-V2 nanoparticles will be evaluated in mice by intramuscular route as well as transcutaneous (skin) route using heat labile enterotoxin as an adjuvant. The immune responses will be quantified for V2- ?4?7 interfering antibodies, virus neutralizing antibodies and transmission-blocking antibodies. The best V2 variants down-selected from these assays will then be tested in the rabbit model. A novel HIV-1 transmission assay will be developed, which will precisely quantify the number of virus genomes that cross the host membrane after a few minutes of exposure to CD4+, ?4?7+ and CCR5+ T cells. Based on real-time PCR, this assay would be extremely sensitive, rapid, and high throughput. Using this assay, V2 variants that can induce antibodies which block virus entry by interfering with the initial virus-host interactions will be selected. These might potentially serve as preventative HIV-1 vaccine candidates for further studies in nonhuman primates and clinical trials.